Limitations in current treatment options for many congenital and acquired diseases in humans, including birth defects, cancer, degenerative disorders and diabetes, highlight the need for the development of novel approaches for regenerative medicine to dramatically improve tissue repair. The pluripotent nature of mammalian embryonic stem cells (ESCs) makes them a convenient model for studying aspects of early development and an invaluable starting point for deriving numerous therapeutically relevant cells for regenerative medicine. Despite the remarkable progress made in deciphering mechanisms driving ESC pluripotency, fundamental gaps remain in understanding how human embryonic stem cells (hESCs) regulate the pluripotent state. If we are to use hESCs as a high-fidelity model for embryonic development, and if we wish to improve outcomes of hESC differentiation and the fidelity of cellular reprogramming to pluripotency, then it is imperative that we understand how hESCs fit into the paradigm of mammalian embryonic development. In this project we seek to answer some of these fundamental questions, by characterizing and validating a potential alternative pluripotency state. Our paradigm-shifting hypothesis stems from our unexpected discovery that the honey bee queen-maker protein, Royalactin, and its structural analog in mammals, Regina, have unexpected robust pluripotency maintenance effects in mammalian stem cells. We hypothesize that Regina/Royalactin stabilize and capture a pivotal pluripotent state distinct from the existing pre- (nave) and post- (primed) implantation associated stem cell states. I outline here a plan to molecularly characterize this novel cellular metastable state with 3 specific aims. In Aim 1, we will Isolate and characterize the composition and activity of the receptor complex(es) in ESCs. We hypothesize that Regina/Royalactin, as secreted molecules, likely directly interact with a receptor partner on the membrane of responsive cells to affect gene expression and subsequent cellular behavior. We will identify the receptor(s) through multiple high throughput forward genetics and proteomic strategies. In Aim 2, we will derive and maintain murine and human ESCs to functionally demonstrate that the Regina/Royalactin-mediated state of pluripotency can be related back to the signaling pathways involved in lineage specification and maintenance in the embryo itself. Establishment of a new distinct stage of mammalian pluripotency will be an important advance in our understanding of early lineage commitment. Lastly, in Aim 3 we will elucidate and characterize the critical mechanisms that interface between Regina/Royalactin and downstream epigenetic and transcriptomic events. The genome-wide analyses will be compared to current established conditions to determine whether genetic and epigenetic instability of the ESCs, associated with impaired developmental potential, exists. Taken together, our data will directly establish how a novel endogenous mammalian pluripotency factor instigates fate decisions in ESCs, and provide a new platform to study the principles governing cell potency, epigenetic regulation, and the mechanisms that regulate developmental processes in nave pluripotent stem cells.